This invention relates to fluorometers and, more particularly, it concerns a low-cost and yet highly effective optical system for a dual channel, ultraviolet-visible, fluorometer in an immunoassay instrument and an associated method of fluorescence spectroscopy.
Fluorometers have gained wide acceptance for clinical analysis of blood and other biological fluids. Conventionally, fluorometers employ an optical system to subject a fluid sample, or a sample containing a fluorescent dye or tag material, to light energy at a first wavelength and cause emission of fluorescent light at a longer wavelength from the sample. The intensity of fluorescent emission is indicative of the presence or quantity of a substance in the sample under investigation. Because the amount of light absorbed and emitted by such biological fluid samples is low, conventional fluorometers are equipped with either one or both of a high output ultraviolet light source and a photomultiplier tube in order to achieve reliable test results.
High output ultraviolet light sources such as xenon arc lamps or lasers are not only expensive, but also suffer from drawbacks such as producing excessive heat, causing irreversible damage to specimens, creating noise, bleaching fluorescent tag materials, and requiring complex and expensive control systems. While it is known in the art to use a less expensive, relatively low ultraviolet output, broadband light source, such as a tungsten halogen lamp, and filter the lamp output with an ultraviolet transmitting bandpass filter, the resulting filtered radiation is of such a low level that the fluorescent light emitted by the sample is difficult to detect Heretofore, the difficulty of fluorescent light detection has been addressed exclusively by the use of an extremely sensitive photomultiplier tube to detect the low levels of sample emitted fluorescence. While providing for radiation detection even at the photon counting level, photomultiplier tubes are expensive and fragile and necessitate relatively complex control circuitry
In light of the foregoing, there is a need for an improved optical fluorometer system which provides the desired analysis without requiring the use of high ultraviolet output light sources and photomultipliers. cl SUMMARY OF THE INVENTION
In accordance with the present invention, an optical system for a fluorometer is provided by which reliable dual channel fluoroanalysis is effected using low cost components.
In a preferred embodiment of the present invention, a low cost and yet highly effective dual channel fluorometer, having excitation and emission branches, incorporates a relatively low ultraviolet output, tungsten halogen excitation source together with solid state photodetectors to detect the low levels of sample emitted light encountered in fluoroanalysis. The attainment of reliable results with such components is made possible by incorporating optics in the excitation and emission branches that are approximately 90 percent transmissive in the ultraviolet region to maximize throughput and by using solid state circuitry together with a filter wheel having both light blocking and light passing regions in a manner which fully accounts for dark signals associated with the solid state photodetectors and amplifiers.
In the practice of the present invention, a sample holder or assay element containing a biological fluid such as blood serum is placed above a read port of the present optical system, illumination provided by the tungsten halogen source and filtered through an excitation branch bandpass filter is focused on the front surface of the sample holder so as to cause the particular constituent under study, or a fluorescent dye or tag material in the sample, to fluoresce. The emitted fluorescence is collected and directed through an emission branch bandpass filter and focused on a photodetector. The present optical system also includes a reference photodetector for receiving illumination from the tungsten halogen source and providing a signal which is used to compensate for variations in source output.
The excitation and emission bandpass filters are carried on a filter wheel as a diametrically opposed matched pair of filters. The filter wheel also includes a pair of diametrically opposed opaque surfaces With the filter wheel in one position, the excitation and emission filters of a matched pair of bandpass filters are simultaneously placed along the excitation and emission paths of the system. With the filter wheel in another position, both the excitation and emission branches are simultaneously blocked by the opaque regions for purposes of obtaining photodetector/amplifier dark signals, which indicate component drift.
The output signals from each of the main and reference photodetectors are amplified, converted, digitized, and processed by solid state circuitry to produce a measurement which is indicative of the concentration of the agent under investigation. The processing is accomplished by using an algorithm based on four sequential photodetector signals, namely, a reference photodetector dark signal, a reference photodetector excitation signal, a main photodetector emission signal, and a main photodetector dark signal. The measurement provided by this algorithm is treated differently by the microprocessor based on the particular type of assay element being used.
A principal object of the invention, therefore, is the provision of a low-cost and yet reliable optical system for a multi-channel fluorometer. Another object of the present invention is the provision of such an optical system which is particularly suited for use in an immunoassay instrument. Yet still another object of the present invention is the use of solid state photodetectors and circuitry in a manner which compensates for the noise and dark signals inherent in such photodetector/amplifiers. Other objects and further scope of applicability of the present invention will become apparent from the detailed description to follow, taken in conjunction with the accompanying drawings in which like parts are designated by like reference characters.